The present invention relates to methods for producing crystalline L-aspartic acid from fumaric acid using aspartase.
Japanese Unexamined Patent Publication No. 48-56618 discloses a method for depositing and recovering D,L-aspartic acid by adding fumaric acid to a disodium D,L-aspartate solution. In this method, disodium fumarate and greatly excessive ammonia are chemically reacted to generate D,L-aspartic acid. After removal of excessive ammonia, fumaric acid is added to the reaction solution to thereby deposit the D,L-aspartic acid, which is then separated from the solution.
In this case, the solution to which fumaric acid is added is a disodium D,L-aspartate solution, and the filtrate obtained by adding fumaric acid to this disodium D,L-aspartate solution and then separating D,L-aspartic acid therefrom is a disodium fumarate solution. It is disclosed that the same reaction is repeated using this filtrate after addition of greatly excessive ammonia in relation to the amount of fumaric acid.
Usually, when L-aspartic acid is produced from diammonium fumarate using an enzyme, the amount of ammonia required is at least 1 time the amount of the raw material fumaric acid in terms of mole. In order to incline the equilibrium of reaction toward L-aspartic acid, usually ammonia is used 2 to 2.3 times the amount of fumaric acid in terms of mole. The optimum pH of aspartase, an enzyme which catalyzes this reaction, is around 8.3. In a pH range which is much higher than this value, various problems occur such as decrease of the enzyme activity or denaturation of the enzyme. Although greatly excessive ammonia is added to the disodium fumarate solution and used in repeated reactions in the method of Japanese Unexamined Patent Publication No. 48-56618, it is not possible to use greatly excessive ammonia in an aspartase enzyme reaction.
The pH of disodium fumarate solution (1.72 mol/l) is 8.4. However, when equimolar ammonia is added thereto, the pH of the resultant solution is 12.1 at 30xc2x0 C. Aspartase will be denatured at such a pH level. Thus, ammonia cannot be used in enzyme reactions using aspartase.
Japanese Patent No. 2524306 discloses a method for depositing and recovering L-aspartic acid by adding fumaric acid to a monoammonium L-aspartate solution. In this method, a diammonium fumarate solution is converted into a monoammonium L-aspartate solution by the action of aspartase, and then fumaric acid is added thereto to deposit L-aspartic acid. After separation of the crystals, ammonia is added to the filtrate, which is then recycled in the subsequent reaction.
In the above method, a salt exchange reaction between L-aspartic acid and fumaric acid is performed by adding fumaric acid to the monoammonium L-aspartate solution under heterogeneous conditions in which fumaric acid crystals and/or L-aspartic acid crystals are constantly present. Since dissolution of fumaric acid and crystallization of L-aspartic acid occur simultaneously in this method, when L-aspartic acid is deposited as crystals, these crystals grow using undissolved fumaric acid crystals as a crystal nucleus. This has caused a problem that the mixed fumaric acid decreases the purity of the resultant L-aspartic acid. Furthermore, since fumaric acid is mixed in the resultant crystals as a crystal nucleus, it cannot be removed efficiently even if various washing operations such as resuspension of the crystals are carried out. In addition, the crystals deposited by this method are extremely small (several micrometers in size) and thus difficult to handle.
As described above, in the method for crystallizing L-aspartic acid by adding fumaric acid to an ammonium salt of L-aspartic acid, no effective method has been found to date in which L-aspartic acid, crystals are deposited from a reaction solution after the solution has been made completely homogeneous.
It is an object of the present invention to solve the problems described above and to provide a method for producing crystalline L-aspartic acid of higher purity using aspartase.
As a result of intensive and extensive researches toward the solution of the above problems, the present inventors have found that the purity of resultant L-aspartic acid crystals can be improved if fumaric acid is added to a preheated ammonium L-aspartate solution and once dissolved homogeneously prior to the deposition of L-aspartic acid.
Briefly, an ammonium fumarate solution is converted into an ammonium L-aspartate solution by the action of aspartase. This solution is heated to 50 to 130xc2x0 C. Fumaric acid is added thereto in an amount 0.4 to 0.8 times the total amount of fumarate and L-aspartate contained therein in terms of mole, and agitated. Then, the fumaric acid is immediately dissolved and a homogeneous, crystal-free solution can be obtained. When this solution which has once become homogeneous is left standing or cooled, it is possible to deposit L-aspartic acid crystals alone. As to the crystal form, it is also possible to obtain needle-like crystals 100-1000 xcexcm in average length by the above procedures.
Thus, if the ammonium L-aspartate solution is preheated, fumaric acid added thereto is immediately dissolved to yield a homogeneous solution. As a result, the contamination of L-aspartic acid crystals with fumaric acid crystals can be prevented.
The inventors have also found that the purity of resultant L-aspartic acid crystals can be improved if, after the addition of fumaric acid to an ammonium L-aspartate solution, L-aspartic acid is deposited by cooling the solution at a rate of 0.1 to 5xc2x0 C. per minute.
It was found that highly pure L-aspartic acid crystals can be obtained by those methods. Thus, the present invention has been achieved.
The present invention encompasses the following inventions.
(1) A method for producing L-aspartic acid comprising treating an diammonium fumarate solution with aspartase to generate an ammonium L-aspartate solution; adding fumaric acid to the solution; and then crystallizing L-aspartic acid from said solution, wherein fumaric acid is added to the ammonium L-aspartate solution after the solution has been heated to 50 to 130xc2x0 C. in an amount 0.4 to 0.8 times the total amount of fumarate and the L-aspartate contained therein in terms of mole, and the resultant mixture is once turned into a homogeneous solution by applying thereto a shearing force, and then L-aspartic acid is deposited therefrom.
(2) The method of (1) above, wherein the temperature of resultant suspension containing L-aspartic acid crystals is in the range from 25 to 100xc2x0 C. when the deposited L-aspartic acid is separated therefrom.
(3) The method of (1) above, wherein the homogeneous solution is retained at 50 to 130xc2x0 C. for 0.1 second to 1 hour.
(4) The method of (1) above, wherein moisture-containing fumaric acid crystals and the ammonium L-aspartate solution are mixed continuously.
(5) The method of (1) above, wherein the solution is cooled at a rate of 0.1-5xc2x0 C./min from the temperature at which fumaric acid is added thereto to the temperature at which crystallized L-aspartic acid is separated therefrom, to thereby deposit L-aspartic acid.
(6) The method of (5) above, wherein the cooling is performed by evaporating water under reduced pressure; condensing the evaporated water by cooling through a condenser; and returning the condensed water to a reactor for L-aspartic acid crystallization or removing the condensed water.
(7) The method of (6) above, wherein pressure reduction at the time of cooling under reduced pressure is performed at a rate of 1-20 torr/min from a range of pressure 10-200 torr higher than the vapor pressure at which the solution to be cooled begins to boil.
(8) The method of (1) above, wherein the crystallizing step is performed by a continuous method.
(9) The method of (1) above, wherein a said ammonium fumarate solution is prepared from a mother liquor from which L-aspartic acid crystals have been removed.
(10) A method for producing L-aspartic acid comprising treating an diammonium fumarate solution with aspartase to generate an ammonium L-aspartate solution; adding fumaric acid to the solution; and then crystallizing L-aspartic acid from the solution, wherein the solution is cooled at a rate of 0.1-5xc2x0 C./min from the temperature at which fumaric acid is added thereto to the temperature at which crystallized L-aspartic acid is separated therefrom, to thereby deposit L-aspartic acid.
(11) The method of (10) above, wherein the solution from which L-aspartic acid is deposited is a homogeneous solution.
(12) The method of (10) above, wherein the cooling is performed by evaporating water under reduced pressure; condensing the evaporated water by cooling through a condenser; and returning the condensed water to a reactor for L-aspartic acid crystallization or removing the condensed water.
(13) The method of (12) above, wherein pressure reduction at the time of cooling under reduced pressure is performed at a rate of 1-20 torr/min from a range of pressure 10-200 torr higher than the vapor pressure at which the solution to be cooled begins to boil.
(14) The method of (10) above, wherein a said ammonium fumarate solution is prepared from a mother liquor from which L-aspartic acid crystals have been removed.
The first invention of the present application is a method for producing L-aspartic acid comprising treating an diammonium fumarate solution with aspartase to generate an ammonium L-aspartate solution; adding fumaric acid to the solution; and then crystallizing L-aspartic acid from said solution, wherein fumaric acid is added to the ammonium L-aspartate solution after the solution has been heated to 50 to 130xc2x0 C. in an amount 0.4 to 0.8 times the total amount of fumarate and the L-aspartate contained therein in terms of mole, and the resultant mixture is once turned into a homogeneous solution by applying thereto a shearing force, and then L-aspartic acid is deposited therefrom.
The second invention of the present application is a method for producing L-aspartic acid comprising treating an diammonium fumarate solution with aspartase to generate an ammonium L-aspartate solution; adding fumaric acid to the solution; and then crystallizing L-aspartic acid from the solution, wherein the solution is cooled at a rate of 0.1-5xc2x0 C./min from the temperature at which fumaric acid is added thereto to the temperature at which crystallized L-aspartic acid is separated therefrom, to thereby deposit L-aspartic acid.
As to the aspartase used for the present invention, a transformant containing a transferred aspartase gene or such a transformant treated and immobilized may be used.
In the above-described method in which a liquid mixture of diammonium fumarate and ammonium L-aspartate containing 5-25% of fumarate and L-aspartate (as calculated for fumaric acid) is fed to a reactor containing an immobilized aspartase having activity of 250 U/ml or more, the liquid feeding rate is preferably in the range from 2 to 25 in terms of LHSV (liquid hourly space velocity).
In the present specification, the following terms have the following meanings:
1 U means production of 1 xcexcmol of L-aspartic acid/min/ml immobilized enzyme
LHSV (liquid hourly space velocity) means liquid fed (ml)/catalyst packed (ml)/hr
Specific examples of immobilized aspartases useful in the present invention include one which is prepared by immobilizing cells or a material obtained from treated cells on an ion exchange resin as a carrier by adsorption or coating with a polymer.
More specifically, an immobilized aspartase prepared as described below may be used. Briefly, a spherical styrene-divinylbenzene copolymer ion exchange resin is selected as an immobilization carrier. A polymer represented by formula (I): 
(wherein Y is a direct linkage or a divalent group represented by one of the formulas below; R1 and R2 are independently hydrogen or an organic residue; Xxe2x8ax96 is an anion; and n is an integer from 100 to 5000) 
is mixed with cells or a material obtained from treated cells. Then, this mixture is coated on the surface of the spherical styrene-divinylbenzene copolymer ion exchange resin for immobilization.
In formula (I) above, specific examples of the organic residue represented by R1 or R2 include alkyl groups with 10 or less carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl. Particularly preferable is methyl. Further, an organic residue having a substituent such as halogen or hydroxyl may also be used. Specific examples of such organic residues include 4-chloro-2,2-dimethylpentyl, 3-ethyl-2,5-dichloroheptyl and 2-hydroxy-3,5-dimethylnonyl. Preferably, 3-chloro-2-hydroxypropyl may be used. As to the anion, a halogen ion such as Fxe2x88x92, Clxe2x88x92, Brxe2x88x92 and Ixe2x88x92 may be used, for example.
This specification includes part or all of the contents as disclosed in the specifications and/or drawings of Japanese Patent Application Nos. 10-278571 and 10-278579, which are priority documents of the present application.